This application proposes to use our model of HIV infection, synaptodendritic damage, and cognitive impairment in mice to address the objectives of RFA-MH-14-170 on establishment and persistence of HIV CNS reservoirs during ART and testing new strategies for mitigation of neurocognitive disease. We focus on mild HAND, neurocognitive impairment (NCI) less severe than dementia, termed HIV-NCI. We have shown that infection of conventional mice with a mouse-tropic chimeric HIV, EcoHIV, reproduces human HIV-NCI in chronic systemic infection, induction of immunity to control HIV in the periphery, early HIV neuroinvasion, and manifestation of a HIV-NCI-like symptomatic disease assessed in behavioral tests. The overall hypothesis of this project is that the biology of persistent HIV infection in CNS in the setting of suppressive ART and restored immunity differs fundamentally from that in peripheral tissues, thereby permitting continuing brain disease and necessitating novel strategies for disease control. We propose that HIV persists in CNS in a biologically active form in long-lived cells; that virus expression and secretion of neuropathogenic products persist despite antiviral drugs and anti-HIV immune responses. The hypothesis will be tested in EcoHIV infected mice using cognitive impairment in behavioral tests to assess the physiological relevance of our studies. Antiretroviral treatment, including CNS delivery, will be optimized using nanoART technology in collaboration with Dr. H. E. Gendelman. The Specific Aims are: 1) Determine the time frame and mechanisms of establishment of HIV CNS reservoirs relative to induction of NCI and post-exposure ART. Using EcoHIV or EcoHIV-GFP, wildtype or CCL2-KO mice, staggered application of nanoART or CCL2 inhibitor, we will test the dynamics of brain infection and viral genome forms, cell types infected, synaptodendritic damage, and NCI induction related to antiviral immunity, ART timing, brain entry by HIV and by monocytes. 2) Determine the mechanisms of HIV persistence in CNS and NCI progression during chronic infection of mice with functional immunity, suppressed peripheral HIV, and chronic ART. In chronic infection with continuous, interrupted, or peripheral-only ART, test the extent of NCI compared to monocyte CNS entry, intra-CNS virus spread, HIV Tat activity in the brain, LPS- induced surges in virus expression, CD8 T cell infiltration, and synaptodendritic injury at the molecular level. 3) Test proof-of-principle of adjunct strategies for HIV provirus activation and silencing for their effects on HIV brain reservoirs and chronic HIV-NCI in the presence of ART. Provirus will be activated with HDAC inhibitors in use. Provirus silencing will include Type I IFN and Tat immunization. The effects of treatment on HIV expression will be correlated with improvement in synaptodendritic injury and HIV-NCI. Methods currently in use include two complementary behavioral tests, QPCR detection of integrated EcoHIV DNA in mouse tissues, gene expression and pathway analysis, dual staining fluorescence microscopy, and HIV immunization.